Labelled carboxylic acids and their uses in molecular imaging

ABSTRACT

The present invention pertains generally to the field of imaging compounds, and more specifically to certain 2,2-dialkyl radionuclide-labelled carboxylic acids suitable for PEWT, SPECT and/or DNP imaging. Also described are uses of such compounds in the imaging of, inter alia, cancer tumors, metastasis, Alzheimer&#39;s disease and multiple sclerosis.

FIELD OF THE INVENTION

The present invention relates to labelled carboxylic acids and their usein molecular imaging, and in particular to radionuclide-labelledcarboxylic acids that do not bear a hydrogen at the carbon alpha to thecarbonyl group, and to 2,2-dialkyl substituted carboxylic acids having alabel suitable for hyperpolarisation.

BACKGROUND OF THE INVENTION

Positron emission tomography (PET) is routinely used in the clinic forcancer detection, with the majority of all scans performed with theglucose analogue [¹⁸F]2-fluoro-2-deoxyglucose, [¹⁸F]FDG. A number ofclinical studies, however, have shown poor sensitivity for the detectionof certain cancers, for example, prostate cancer, by [¹⁸F]FDG-PET.Prostate cancer is the most prevalent of cancers in the male population,accounting for 40,841 incidents in the UK in 2009.¹ In the USA prostatecancer is the second largest cause of cancer mortality amongst men. Thepoor sensitivity for prostate cancer detection by [¹⁸F]FDG-PET isthought to be a result of low basal glucose metabolism of some prostatetumours² and the high renal clearance of [¹⁸F]FDG, which can often masktumour uptake.³ As a result, other PET tracers have been developed forcancer imaging including as [¹¹C] choline, [¹⁸F]choline and[¹¹C]acetate.

[¹¹C]Acetate was initially developed as a radiotracer to evaluateoxidative metabolism in the myocardium. Following entry into the cell,either by passive diffusion or membrane transport via themonocarboxylate transporters,⁴ [¹¹C]acetate is converted to[¹¹C]acetyl-CoA by acetyl CoA synthetase before its rapid metabolism viathe citric acid cycle to [¹¹C]CO₂.⁵ As well as being a substrate for thecitric acid cycle, acetyl-CoA also enters into the fatty acid synthesispathway, with tumour-associated [¹¹C]acetate accumulation shown toresult from cell membrane incorporation following flux through the fattyacid synthesis pathway.

Experiments carried out by Yoshimoto and co-workers showed that[1-¹⁴C]acetate was incorporated into the lipid soluble fraction, mostlyas phosphatidylcholine and neutral lipids. The accumulation of [1-¹⁴C]acetate was positively correlated with the growth of the tumour cells.Fatty acid synthase (FAS) has been shown to be overexpressed in cancer,accounting for the uptake of acetate into the fatty acid synthesispathway and incorporation into the cell membrane.^(6,7) [¹¹C]Acetate hasshown great promise in imaging prostate cancer, but the short half-lifeof carbon-11 (20.4 min) requires an on-site cyclotron, limiting itswide-spread use. [¹⁸F]Fluoroacetate ([¹⁸F]FAC, 1) has been investigatedas an alternative to [¹¹C]acetate for imaging of prostate cancer. Theradiotracer was introduced by Welch and co-workers.⁸ The advantage ofusing fluorine-18 is its longer half-life of 109.5 min compared to thecarbon-11 half-life of 20.4 min.

The authors in reference 8 investigated the biodistribution of[¹¹C]acetate and [¹⁸F]FAC (1) in Sprague-Dawley rats. They found fairlyrapid clearance of [¹¹C]acetate from most of organs except the pancreasat 1 h, whereas [¹⁸F]FAC clearance was slower from most organs. This isthought to be due the oxidative metabolism of [¹¹C]acetate, releasing[¹¹C]CO₂. The main drawback of [¹⁸F] FAC is its substantial bone uptake,characteristic of radiotracer defluorination.⁹ A comparison with[¹¹C]acetate did describe a sizeable amount of the injected dose of[¹⁸F]FAC was taken up by bones in pigs and less pronounced in monkeys.This unwanted and massive defluorination in pigs results in unspecificintense skeletal activity and imaging during PET, showing this tracer'slimitations for use in higher animals. [¹⁸F]Fluoroacetate is not afunctional analogue of [¹¹C]acetate in normal physiology.⁹ There are anumber of putative routes for defluorination (Scheme 1). Tecle andCasida, for example, found that incubation of [¹³C]fluoroacetate withrat and mouse liver cytosol leads to formation of S-([¹³C]carboxymethyl)glutathione (2) and fluoride ion indicating that the fluoride ion isdisplaced by the glutathione (GSH) via a nucleophilic attack. Once[¹⁸F]FAC enters the citric acid cycle, it is converted into2-fluorocitrate 3. The same authors,¹⁰ together with othergroups,^(11,12) showed that (−)-erythro-2-fluorocitrate is both asubstrate and an inhibitor for aconitase, the latter is responsible fordefluorination. The mechanism of defluorination requires the conversionof 2-fluorocitrate 3 into fluoro-cis-aconitate, which undergoes additionof hydroxide and subsequent loss of fluoride to form4-hydroxy-trans-aconitate 4. Compound 4 binds very tightly to the enzymeand it is responsible for the toxicity of fluoroacetate atpharmacological levels.

Given the inadequate performance of [¹⁸F]FAC as a tracer that can beused to image biology of acetate metabolism preclinically in prostatecancer and beyond, there is an unmet need to provide a stable imagingagent which does not undergo de-labelling to lose its radionuclidelabel.

Dynamic nuclear polarization (DNP) of ¹³C-labeled molecules can increasetheir sensitivity of detection in a solution-state nuclear magneticresonance experiment by >10,000 times.¹³ This dramatic increase insensitivity means that, after intravenous injection, the spatialdistribution of the labelled molecule and its subsequent metabolism canbe imaged in vivo using ¹³C magnetic resonance spectroscopic imaging(MRSI) techniques. Tracking metabolic reactions in vivo by DNP has beenexemplified with hyperpolarised [1-¹³C]pyruvate, whose metabolicproducts, [1-¹³C]lactate, [1-¹³C] alanine, and [¹³C]bicarbonate, havebeen shown to correlate with disease progression and response totherapy.

In tumours, the metabolic fate of [1-¹³C]pyruvate is label exchange to[1-¹³C]lactate, catalysed by lactate dehydrogenase, the final enzyme inthe glycolytic pathway. Since the pyruvate blood-brain barrier (BBB) israte limited,¹⁴ hyperpolarised [1-¹³C]pyruvate may have limited utilityfor determining disease-state under conditions where an intact BBB ispresent, for example, infiltrating gliomas, Alzheimer's disease,nonenhancing multiple sclerosis, and acute stroke. Unsubstituted[1-¹³C]propionate has previously been polarised to high levels by DNP,and its metabolic products imaged in vivo during ischemia.¹⁵

There exists a need to provide imaging agents for DNP that can measuredisease-state in the brain. Moreover, a measure of metabolic flux inpathways other than glycolysis may provide alternate and complementaryprognostic information for diseases in other tissues of the body.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to compositions comprising noveltracers for use in molecular imaging, and to the use of thesecompositions as imaging agents in molecular imaging. In particular, thepresent invention is based on the inventors' insight thatradionuclide-labelled carboxylic acids that are devoid of protons at theposition alpha to the carbonyl group of the carboxylic acid may beuseful as radiotracers that are resistant to the de-labelling pathwaysdescribed above (Scheme 1) and the associated drawbacks, while stillexhibiting the desired uptake profile. Additionally, the presentinvention is based on the insight that such compounds having a labelsuitable for polarization using dynamic nuclear polarisation (DNP) inaddition to, or as an alternative to, a radionuclide label may be usefultracers for spectroscopic imaging.

The compositions of the present invention are generally applicable formolecular imaging of diseases and disorders using PET and/or SPECT(depending on the radionuclide chosen) and/or spectroscopic imaging bymagnetic resonance spectroscopy (MRS) or magnetic resonance imaging(MRI) following polarisation of the molecule by dynamic nuclearpolarisation (DNP) if a suitable isotope, for example, [¹³C] carbonor[¹⁵N] nitrogen, is included. Accordingly, compositions of the inventionmay be used, for example, for the imaging of tumours, metastasis andheart-related diseases and disorders. In particular, compositions of thepresent invention may have utility as tracers for the imaging of tumourswhich have a high fatty acid turnover and/or are hypoxic or for which[¹⁸F]FDG imaging is sub-optimal.

Compositions of the present invention may be useful as tracers in thedetection of acetyl-CoA synthetase (ACSS) activity in, for example,tumour cells. ACSS is expressed in 2 isoforms: ACSS1 & 2. Some tumourcells have a higher expression level of ACSS 1/2 than normal cells andhypoxia increases ACSS activity even more (also free fatty acids).Therefore, tissue of tumours of these types will be enriched for thetracers of the present invention.

Without wishing to be bound to any particular theory, the presentinventors believe that ACSS1/2 activity results in the carboxylic acidmoieties of the tracer compounds of the present invention beingconverted to the corresponding CoA adduct. Further oxidation to CO₂ isnot possible in mammalian cells.

The present inventors have found that increased uptake of compounds ofthe invention may be observed following incubation with L-carnitine. Ascarnitine esters are known to be products of both [¹³C]acetate and[¹³C]propionate catabolism in the heart, without wishing to be bound byany particular theory, the inventors believe that the mechanism oftrapping of compounds of the invention may occur via CoA and carnitineester formation. Transesterification of CoA adduct to a carnitine esteroccurs under the action of carnitine acyl transferases.²⁵ The carnitineadduct is membrane impermeable and may be transported out of tissues viathe human kidney carnitine transporter (hOCTN2), whose transport iscompetitively inhibited by L-carnitine.²⁶ Uptake may work via twodifferent mechanisms: diffusion or possibly by facilitated diffusion. Inaddition to ACSS activity, compositions of the present invention may beuseful as tracers in the detection of cartinie acetyltransferaseactivity and/or activity of the facilitated diffusion transporter in,for example, tumour cells.

In a first aspect, the present invention relates to compositionscomprising a tracer, wherein the tracer is a labelled carboxylic acid,or the corresponding carboxylate anion thereof, that does not bear ahydrogen at its alpha carbon. These compounds are able to form thedesired CoA- and carnitine adducts but, owing to the absence of alphaprotons, are not metabolised and are therefore more stable than, forexample, [¹⁸F]FAC. The labeled carboxylic acids described herein areshort alkyl chain carboxylic acids, for example,2,2-disubstituted-acetic, -propanoic, -butanoic or -pentanoic acids. Itwill be appreciated that, as described herein, these acidic backbonesmay be substituted in addition to being labelled.

Accordingly, described herein are radionuclide-labelled carboxylic acidsthat are compounds of formula (I):

wherein Q is a suitable radionuclide; C^(L) is [¹¹C], [¹²C] or [¹³C],optionally C^(L) is [¹²C] or [¹³C], preferably [¹²C]; n may be 0, 1, 2,or 3, for example, n may be 0, 1, or 2, preferably 1 or 2; R¹ and R² areinductively electron-donating substituents; and R³ and R⁴ areindependently H or F.

A value of n>0 may be advantageous and result in compounds of theinvention that are even less reactive towards GSH due to the increaseddistance of the activating carboxylic acid/carboxylate group.

In a first aspect, the present invention provides a compositioncomprising a tracer, wherein the tracer is a labelled2,2-di-C₁₋₄-alkylpropanoic acid, 2,2-di-C₁₋₄-alkylbutanoic acid,2,2-di-C₁₋₄-alkylpentanoic acid, or the corresponding carboxylate,wherein the tracer is labelled with:

a radionuclide and/or

a label suitable for polarisation using dynamic nuclear polarisation(DNP).

Suitably, the tracer comprises a radionuclide. For example, the tracermay be a radionuclide-labelled carboxylic acid or the correspondingcarboxylate, wherein the radionuclide-labelled carboxylic acid is acompound of formula (I):

wherein

Q is a suitable radionuclide;

C^(L) is selected from [¹¹C]carbon, [¹²C]carbon or [¹³C]carbon;

n is 1, 2, or 3;

R¹ and R² are independently C₁₋₄-alkyl; and

R³ and R⁴ are independently H or F.

Preferably, R³ and R⁴ are both hydrogen.

Preferably, n is 1 or 2, more preferably, n is 1.

In preferred compounds, R¹ and R² are both methyl.

In preferred compounds of the invention, R¹ and R² are both methyl, n is1, and R³ and R⁴ are both H.

Suitable radionuclides are known in the art and include [¹¹C]carbon,[¹⁸F]fluorine, [¹³N]nitrogen, [¹⁵O]oxygen, [¹⁶Br]bromine, [¹²³I]iodine,[¹²⁴I]iodine, and [¹²⁵I]iodine. An especially preferred radionuclide is[¹⁸F]fluorine.

C^(L) is a carbon isotope selected from [¹¹C]carbon, [¹²C]carbon and[¹³C]carbon. In some embodiments, C^(L) may be selected from[¹²C]carbon, [¹²C]carbon. For example, C^(L) may be [¹²C]carbon. Inother embodiments, C^(L) may be [¹³C]carbon. In these cases, the[¹³C]carbon is a label suitable for hyperpolarisation using DNP.

The tracer may be a labelled carboxylic acid or the correspondingcarboxylate of formula (Ia):

wherein:

R¹ and R² are C₁₋₄alkyl;

C^(L) is [¹¹C]carbon, [¹²C]carbon, or [¹³C]carbon;

W is R⁵ or Q;

R³ is H, F, Cl, Br, I, or NH₂; and

Q is a radionuclide.

In some embodiments, W is Q. In other embodiments, W is R⁵.

In preferred compounds, R¹ and R² are both methyl.

In some embodiments, the tracer is a [1-¹³C]2,2-dimethylpropanoic acid,wherein the three position is substituted with F, [¹⁸F]F, Cl, Br,[⁷⁶Br]Br, I, [¹²³I]I, [¹²⁴I]I, [¹²⁵I]I, or NH₂, optionally with F,[¹⁸F]F, Cl, Br, [⁷⁶Br]Br, [¹²³I]I, [¹²⁴I]I, or [¹²⁵I]I.

In preferred compounds of the invention, R¹ and R² are both methyl, n is1, and R³ and R⁴ are both H. In some preferred embodiments, the compoundis selected from:

[¹⁸F]3-fluoro-2,2-dimethylpropanonic acid, referred to herein as[¹⁸F]FDMP and as [¹⁸F]FPIA;

[1-¹³C][¹⁸F]3-fluoro-2,2-dimethylpropanonic acid, referred to herein as[1-¹³C][¹⁸F]FDMP and as [1-¹³C][¹⁸F]FPIA; and

[1-¹³C]3-fluoro-2,2-dimethylpropanonic acid, referred to herein as[1-¹³C]FDMP and as [1-¹³C]FPIA.

In a further aspect, the present invention provides use of compositionsof the present invention as imaging agents. Compositions comprisingtracers labelled with radionuclides as described herein are suitable forimaging techniques that detect gamma rays.

In a further aspect, the present invention provides compositions of thepresent invention for use in a method of imaging for diagnosing acondition in a subject, wherein the method comprises:

-   -   (i) administering the composition to the subject;    -   (ii) detecting gamma rays emitted, either directly or        indirectly, by the tracer;    -   (iii) acquiring at least one image associated with the gamma        rays emitted by the tracer; and    -   (iv) diagnosing the condition in the subject using the image.

In some methods of the present invention, the composition may bepre-administered. Accordingly, in a further aspect, the presentinvention provides compositions according to the present invention foruse in a method of imaging for diagnosing a condition in a subject,wherein the subject has been pre-administered with a compositionaccording to the present invention, wherein the method comprises:

-   -   (i) detecting gamma rays emitted, either directly or indirectly,        by the tracer;    -   (ii) acquiring at least one image associated with the gamma rays        emitted by the tracer; and    -   (iii) diagnosing the condition in the subject using the image.

In a further aspect, the present invention provides methods for imaginga condition in a subject using a composition according to the presentinvention, the methods comprising the steps of:

-   -   (i) administering a composition comprising a tracer according to        the present invention to the subject;    -   (ii) detecting gamma rays emitted by the tracer; and    -   (iii) acquiring at least one image associated with the gamma        rays emitted by the tracer.

The method may then further comprise the step(s) of diagnosing thecondition in the subject using the image and/or comparing the image witha previously obtained image to monitor progression of the conditionand/or response to therapy.

In a further aspect, the present invention provides methods fordetecting a condition in a subject using compositions according to thepresent invention, the subject having been pre-administered with acomposition according to the present invention, the methods comprisingthe steps of:

-   -   (i) detecting gamma rays emitted, either directly or indirectly,        by the tracer; and    -   (ii) evaluating the condition based on the detection of the        gamma rays emitted by the tracer.

Also described herein are compositions comprising a tracer suitable forspectroscopic imaging by MRS or MRI following polarisation of themolecule by DNP, wherein the tracer is a carboxylic acid or thecorresponding carboxylate that does not bear a hydrogen at its alphacarbon, the alpha carbon being substituted with at least twosubstituents, wherein the tracer comprises a [¹³C] or [¹⁵N] label. Insome embodiments, the carboxylic acid is a substituted propanoic acid,for example, [1¹³C]-2,2-dimethylpropanoic acid. The three position maybe optionally substituted with F, [¹⁸F]F, Cl, Br, [⁷⁶Br]Br, [¹²³I]I,[¹²⁴I]I, or [¹²⁵I]I, preferably F or [¹⁸F]F.

It will be appreciated that imaging may use one or more imagingtechniques, for example, the imaging may be PET/SPECT, PET/SPECT and DNPor DNP alone.

Accordingly, the present invention further provides a compositioncomprising a tracer, wherein the tracer is a labelled2,2-di-C₁₋₄-alkylpropanoic acid, or the corresponding carboxylate,wherein the tracer is labelled with a radionuclide and/or a labelsuitable for polarisation using dynamic nuclear polarisation (DNP),wherein the tracer is a labelled carboxylic acid or the correspondingcarboxylate of formula (Ia):

wherein:

R¹ and R² are C₁₋₄alkyl;

C^(L) is [¹¹C]carbon, [¹²C]carbon, or [¹³C]carbon;

W is R⁵ or Q;

R⁵ is H, F, Cl, Br, I, NH₂; and

Q is a radionuclide.

In some embodiments, C^(L) is [¹³C]carbon.

Accordingly, in a further aspect, the present invention provides acomposition comprising a tracer suitable for MRS or MRI followingpolarisation of the molecule by DNP for use in a method of imaging fordiagnosing a condition in a subject, wherein the method comprises:

-   -   (i) hyperpolarising the tracer compound by DNP;    -   (ii) administering the composition to the subject;    -   (iii) collecting MRS or MRI data associated with the        hyperpolarized tracer compound;    -   (iv) acquiring at least one image by MRS or MRI; and    -   (v) diagnosing the condition in the subject using the image.

In yet a further aspect the present invention provides compositionscomprising a tracer suitable for spectroscopic imaging by MRS or MRIfollowing polarisation of the molecule by DNP for use in a method ofimaging for diagnosing a condition in a subject, wherein the subject hasbeen pre-administered with a composition, the composition having beenhyperpolarized by DNP, wherein the method comprises:

-   -   (i) collecting MRS or MRI data associated with the        hyperpolarized tracer compound;    -   (ii) acquiring at least one image by MRS or MRI; and    -   (iii) diagnosing the condition in the subject using the image.

In any of the imaging methods of the invention, a second imagingtechnique or step may be used, and for compounds of the inventioncomprising both a radionuclide and a label suitable forhyperpolarisation, a combination of PET and/or SPECT with MRS or MRIfollowing hyperpolarision of the tracer compound by DNP. In any of theimaging methods of the invention, the subject may be human or animal.

In a further aspect the present invention provides a method ofsynthesising a composition comprising a compound of formula (II)

the method comprising the step of treating a compound of formula (III)

with a reagent comprising a radionuclide nucleophile to give a compoundof formula (IV)

wherein Q is a radionuclide or group comprising a radionuclide; C^(L) isselected from [¹²C]carbon or [¹³C]carbon, preferably [¹²C]carbon; andR¹, R² and R³ are independently C₁₋₄alkyl.

In a further aspect, the present invention provides a kit for thepreparation of a radionuclide-labelled imaging agent, the kit comprisinga compound of formula (III) and instructions for a method according tothe present invention.

Embodiments of the present invention will now be described by way ofexample and not limitation with reference to the accompanying figures.However various further aspects and embodiments of the present inventionwill be apparent to those skilled in the art in view of the presentdisclosure.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. PET imaging with [¹⁸F]FDMP in healthy BALB/c mice.Representative sagittal CT (left), PET (middle), and PET-CT (right)images (30-60 min summed-activity) for [¹⁸F]FDMP. Key organs areidentified. Abbreviations: B, brain; H, heart; L, liver; K, kidney; I,intestines.

FIG. 2. Biodistribution time course of [¹⁸F]FDMP in EMT6xenograft-bearing BALB mice. Approximately 3.7 MBq of [¹⁸F]FDMP wasadministered i.v. into anaesthetized animals prior to sacrifice atindicated time points. Tissues were excised, weighed and counted, withcounts normalized to injected dose/g wet weight tissue. Mean values(n=3) and SD are shown.

FIG. 3. Comparison between EMT6 tumour xenograft and background tissue[¹⁸F]FDMP radioactivity. Values were obtained from time coursebiodistribution studies. Mean values and SD are shown (n=3). ***,P<0.005 tumour/muscle ratio; ###, P<0.005 tumour/blood ratio.

FIG. 4. Time course of [¹⁸F]FDMP uptake in vitro in EMT6 murine breastadenocarcinoma cells. Mean values and SD are shown (n=3).

FIG. 5. Representative radio-HPLC analysis of mouse tissue extracts.Tissues were obtained 30 min after i.v. injection of [¹⁸F]FDMP intonon-tumour-bearing BALB/c mice and compared to injection-ready [¹⁸F]FDMPas a standard. Figure table—urine metabolite analysis. Percentage urineradioactivity of [¹⁸F]FDMP (peak 2; retention time 7.8 min) and unknownmetabolites (peak 1, retention time 6.5 min; and peak 3, retention time9.75 min) are shown. Mean±SD (n=3 mice).

FIG. 6. Time course of [¹⁸F]FDMP uptake in vivo in EMT6 murine breastadenocarcinoma xenografts in comparison to normal healthy tissues. Meanvalues and SD are shown (n=3). 30 to 60 min cumulative images of thedynamic data were employed to define 3-dimensional (3D) regions ofinterest (ROIs). The count densities were averaged for all ROIs at eachtime point to obtain a time versus radioactivity curve (TAC), normalisedfor injected dose.

FIG. 7. Dynamic [¹⁸F]FDG- and [¹⁸F]FDMP-PET image analysis. (A)Representative coronal PET-CT images (30-60 minutes of summed activity)for [¹⁸F]FDMP and [¹⁸F]FDG. (B) Representative axial PET-CT images ofEMT6 tumour-bearing mice (30-60 minutes of summed activity) for[¹⁸F]FDMP and [¹⁸F]FDG. White arrowheads indicate the tumour, identifiedfrom the CT image. (C) EMT6 tumour time versus radioactivity curve (TAC)obtained from 60-minute dynamic PET imaging. Mean±SD (n=5 mice pergroup). (D & E) Semi-quantitative imaging variables extracted from theTAC. (D) Average tumour-associated counts at 60 min, normalised forinjected dose. (E) Area under the tumour TAC. Mean±SD (n=4 mice pergroup).

FIG. 8. [¹⁸F]FDMP and [¹⁸F]FDG biodistribution in U87 humanglioblastoma-bearing BALB/c nude mice. Approximately 3.7 MBq ofradiotracer was administered i.v. into anaesthetized animals prior tosacrifice at 60 min post injection. Tissues were excised, weighed andcounted, with counts normalized to injected dose/g wet weight tissue.Mean values (n=3) and SD are shown. *, P<0.05; *****, P<0.00005.Abbreviations: NS, not significant.

FIG. 9. [¹⁸F]FDMP uptake and metabolism in cancer cells. (A) Effect ofexogenous 10 μM carnitine on [¹⁸F]FDMP uptake, trapping and retention inEMT6 cells. (B) Intracellular accumulation of [¹⁹F]FDMP and [¹⁹F]FAC inhuman breast adenocarcinoma BT474 cells at 24 h as analyzed by LC-MS.Mean±SD (n=3).

FIG. 10. Effect of exogenous [¹⁹F]FDMP and [¹⁹F]FAC on intracellularmetabolite concentrations of fluorocitrate (A), citrate (B),cis-aconitate (C) and α-ketoglutarate pools (D) as analyzed by LC-MS.AU, arbitrary units.

FIG. 11. Comparison between [¹⁸F]FDMP uptake in murine EMT6 and humanBT474 breast tumours 60 min post radiotracer injection (n=4 mice pergroup).

FIG. 12. Prostate tumour imaging with [¹⁸F]FDMP and [¹⁸F]FDG-PET. (A)Representative axial and coronal PET images of DU145 tumour-bearing mice(50-60 minutes of summed activity) for [¹⁸F]FDMP and [¹⁸F]FDG. (B)Semi-quantitative tumour uptake values for DU145 and EMT6 tumours,extracted from the PET images and normalized to whole-bodyradioactivity. Mean±SD (n=4 mice per group). *** P<0.001; N.S., notsignificant.

FIG. 13. [¹⁸F]FDMP and [¹⁸F]FDG uptake in an aseptic inflammation model.(A) Representative coronal PET images (30-60 summed frames) for[¹⁸F]FDMP and [¹⁸F]FDG. Turpentine-induced inflammatory tissue iscircled in white. (B) [¹⁸F]FDMP and [¹⁸F]FDG biodistribution of theturpentine-treated and control, untreated posterior thigh muscle.Mean±SD (n=3-4). *** P<0.001. (C) Immunohistochemistry analysis by H&Estaining in control and turpentine-treated muscle. Representativephotographic images of H&E-stained sections were acquired at 200×magnification. Scale bar=100 μm.

FIG. 14. [¹⁸F]FDMP and [¹⁸F]FDG uptake in an orthotopic U87 brain tumourmodel. PET images were acquired 15-30 min and 45-60 min post radiotracerinjection 6 weeks after intracranial injection of U87 glioma cells.White arrows indicate the tumour.

DETAILED DESCRIPTION

Tracers

At their most general, the tracer compounds of the invention arelabelled carboxylic acids that do not bear a hydrogen at the positionalpha to the carboxylic acid carbonyl, that is, at the 2 position.Accordingly, compounds of the invention are labelled 2,2-disubstitutedcarboxylic acids, wherein the two 2-substitutents may be referred to asR¹ and R². The carboxylic acids of the invention are short alkyl chain(e.g. C₃₋₅) carboxylic acids, for example and not by way of limitation,2,2-disubstituted propanoic, butanoic or pentanoic acids.

It will be understood that the carboxylic acid moieties of compounds ofthe present invention will be prone to deprotonation and all referencesto carboxylic acid compounds herein include the correspondingcarboxylate anion.

Preferred substituents include inductively electron-donatingsubstituents, for example, C₁₋₄alkyl substituents, wherein the C₁₋₄alkylmay be linear or branched, and saturated or unsaturated. Examples ofsuitable C₁₋₄alkyl substituents may include, but are not limited to,methyl, ethyl, vinyl, n-propyl, i-propyl, allyl, n-butyl, i-butyl,s-butyl, and t-butyl. For example, in some embodiments, R¹ and R² areselected from the following combinations:

R¹ R² Me Me Me tBu nBu tBu Me iPr Et nBu Me Allyl nBu nBu iBu Allyl

Described herein are tracer compounds of formula (I):

In compounds of formula (I), Q is a label, preferably a radionuclidesuitable for use in PET or SPECT methods. Details of suitable labels foruse in PET or SPECT methods are provided below. In compounds of formula(I), n may be 0, 1, 2, or 3, preferably 1, 2, or 3, more preferably 1 or2, especially preferably 1.

In compounds of formula (I), R¹ and R² are inductively electron-donatingsubstituents, preferably alkyl groups as detailed above, more preferablymethyl groups.

In compounds of formula (I), R³ and R⁴ are independently hydrogen orfluoride, preferably hydrogen.

Optionally, a carbon isotope other than the most naturally-abundantcarbon isotope, [¹²C]carbon, may be incorporated at 1-C. For example,incorporation of a [¹³C]carbon at 1-C.

In some preferred embodiments, the tracer compounds of the presentinvention are labelled 2,2-disubstituted short alkyl chain carboxylicacids, for example, propanoic acids, wherein the two 2-substituents areC₁₋₄alkyl. In some embodiments, the compounds are compounds of formula(Ia) comprising a radionuclide and/or a label suitable for imaging byMRS or MRI following hyperpolarization by DNP.

wherein R¹ and R² are C₁₋₄alkyl; C^(L) is [¹²C]carbon, [¹¹C]carbon, or[¹³C]carbon; and W is R⁵ or Q, wherein R⁵ is H, F, Cl, Br, I, or NH₂,preferably H or F, and Q is a radiolabel as defined herein.

In some embodiments, C^(L) is [¹²C] carbon and W is Q.

In some preferred compounds of the invention both of the 2-substituentsare methyl, that is, the compounds are compounds of formula Ib:

wherein C^(L) and W are as defined herein, and the compounds comprise aradionuclide and/or a label suitable for imaging by MRS or MRI followinghyperpolarization by DNP.

The tracer compounds of the present invention comprise a label that isan isotope suitable for detection in medical imaging techniques. In someembodiments, the compounds comprise a radionuclide suitable fordetection PET and/or SPECT imaging techniques. Suitable radionuclidesfor PET are known in the art and include, but are not limited to,[¹¹C]carbon, [¹³N]nitrogen, [¹⁵O]oxygen, [¹⁸F]fluorine, [⁷⁶Br]bromine,and [¹²⁴I]iodine. Suitable radionuclides for SPECT are known in the artand include [¹²³I]iodine, and [¹²⁵I]iodine. Accordingly, in someembodiments of the present invention, the radionuclide is selected from[¹¹C]carbon, [¹³N]nitrogen, [¹⁵O]oxygen, [¹⁸F]fluorine, [⁷⁶Br]bromine,[¹²³I]iodine, [¹²⁴I]iodine, and [¹²⁵I]iodine. In some embodiments, theradionuclide is selected for PET imaging and is selected from[¹¹C]carbon, [¹³N]nitrogen, [¹⁵O]oxygen, [¹⁸F]fluorine, [⁷⁶Br]bromine,and [¹²⁴I]iodine. Preferably, the radionuclide is [¹⁸F]fluorine. In someembodiments, the radionuclide is selected for SPECT imaging and isselected from [¹²³I]iodine and [¹²⁵I]iodine. Additionally oralternatively, the compounds may comprise a label suitable forhyperpolarisation, such that the compound may be used in DNP imagingmethods. Suitable labels include [¹³C] and [¹⁵N]. It will be appreciatedthat where a label has additional valencies, these valencies areoccupied by hydrogen radicals. For example, an [¹⁵N] label is present as[¹⁵N]H₂.

A particularly preferred compound is [¹⁸F]3-fluoro-2,2-dimethylpropanoicacid, which may also be called [¹⁸F]3-fluoro-2,2-dimethylpropionic acidand [¹⁸F]3-fluoranyl-2,2-dimethylpropionic acid, and is also referred toherein as [¹⁸F]FDMP and as [¹⁸F]FPIA ([¹⁸F]fluoropivalic acid).

Uses of the Present Invention

The tracer compounds of the present invention may be used for themolecular imaging of diseases, such as cancer, metastasis, inflammatorydiseases such as multiple sclerosis (MS) and neurodegenerative diseasessuch as Alzheimer's disease, and heart-related diseases and disorders.The applications of the imaging tracers of the present invention includea wide range of imaging and spectroscopic applications that can employthe imaging tracer and/or a further label, for example, in multi-modalimaging studies. As described herein, radio-labelled tracers of thepresent invention are particularly useful for in vivo imagingapplications such as PET and SPECT. This might be useful in a number ofdifferent medical or research applications, for example in the fields ofcancer detection and characterisation, the monitoring of diseaseprogression and treatment effects/outcomes, and in the detection andmonitoring of heart-related diseases and disorders.

The present invention is particularly relevant to nuclear medicineimaging techniques, such as PET, an imaging technique that providesthree-dimensional images by detecting pairs of gamma rays emittedindirectly by a positron-emitting radionuclide introduced into a sampleor subject, and SPECT, an imaging technique that detects gamma raysemitted from a radionuclide to produce a three dimensional image of thedistribution of the radionuclide in a sample or subject.

In some embodiments of the invention, the compounds comprise a ¹⁵N or¹³C label for spectroscopic imaging by MRS or MRI following polarisationof the molecule by DNP. In preferred compounds, the label is ¹³C and ispreferably at the C-1 position of the carboxylic acid lacking a hydrogenat its alpha carbon, that is, the compound may be, for example, a[1-¹³C]-2,2-disubstituted propanoic acid such as [1-¹³C]FDMP.

The tracers of the present invention may be used in methods ofmulti-modal imaging, that is where information or images are derivedfrom two different techniques, either by the detection of the imagingtracer capable of detection using two different techniques as explainedin detail herein or by providing a second label at the site in thebiological system where the tracer becomes localised, most convenientlyby linking or associating the second label with the tracer. Multi-modalstudies will be co-registered and may entail simultaneous imaging withtwo modalities or may need to take place in two steps, but generallyemploy the same sample so that spatial information obtained using thetwo techniques can be compared. Accordingly, in some methods of thepresent invention a second imaging technique is used. Examples ofmulti-modal imaging include PET/CT, SPECT/CT, PET/MR and SPECT/MR.

[¹⁸F]Fluorine labelled compounds of the invention may be especially asadvantageous as, owing to the long half-life of [¹⁸F] fluorine incombination with the enhanced stability to metabolism associated withcompounds of the invention, these compounds may be especially useful inperforming duel-case PET/CT imaging to distinguish betweenbenign/reactive and malignant lymph nodes, for which type of imaging astable tracer with a long half-life is needed.

It may be preferable to incorporate two labels into a compound of thepresent invention, for example, a radionuclide for detection using PETor SPECT, and a label suitable for spectroscopic imaging by MRS or MRIfollowing polarisation of the molecule by DNP, such as a [¹³C]carbon.Examples of suitable compounds could include, for example,[¹⁸F][1-¹³C]-2,2-disubsituted propanoic acids such as [¹⁸F][1-¹³C]FDMP.In methods using compound tracers of this type, the second imagingtechnique includes the step of hyperpolarising the molecule by DNPbefore obtaining the MRS or MRI.

Owing to their stability to metabolism, tracers of the invention may beuseful for multimodal imaging with a time delay between imaging steps.For example, it may be useful to image the tracer at delayed time pointsto improve signal to noise contrast for the imaging of certainconditions and tumours, for example, metastasis. Accordingly, in somemethods of the present invention, the method further comprises obtainingmultiple data sets associated with imaging the tracer, these data setsbeing obtained at spaced time intervals of, for example, between about30 min and 1 h. The compositions and methods of the present inventionmay further have utility in multimodal imaging techniques having a timedelay between imaging sessions, for example in dual-case PET/CTtechniques which feature two imaging sessions over a course of time.

The compositions and methods of the present invention may be used asimaging agents and may useful in a variety of clinical and pre-clinicalsettings. For example, the imaging compositions and methods of imagingof the present invention may be useful at the diagnosis stage and duringtreatment. Imaging may be used to detect the presence of, for exampleand not by way of limitation, a lesion, diseased tissue, a tumour,metastasis or a heart-related condition or disorder and/or to quantifythe size or distribution of the lesion, diseased tissue, tumour,metastasis, a genetic/epigenetic disease of lipid metabolism, orheart-related condition or disorder such as heart disease. Examples ofgenetic/epigenetic diseases of lipid metabolism may include, but are notlimited to, Gaucher disease, Tay-Sachs disease (also known as GM2gangliosidosis or hexosaminidase A deficiency). Other inflammatorydiseases can be detected, for example multiple sclerosis, in addition toneurodegenerative diseases, including Alzheimer's disease.

Methods of the present invention may be used to monitor a condition in asubject after diagnosis to determine and/or monitor disease progression,amelioration, shrinkage, response to treatment, for example, drug orradiation therapy, etc. and to detect any changes in, for example,tumour behaviour. Accordingly, in some embodiments, methods of imagingand methods of imaging for the purposes of diagnosing, evaluating ormonitoring a condition may be repeated more than once to follow theprogress of a condition and/or response to treatment such as drug orradiation therapy, over time.

In some methods of the present invention, the condition is a lesion ordiseased tissue that has a high lipid metabolism compared to levels inhealthy tissue of the same organ or origin, and may, for example, be atumour having increased ACSS activity/expression, in particular,increased ACSS2 activity/expression, increased carnitineacetyltransferase activity/expression, and/or activity of thefacilitated diffusion transporters. The tumour may be a hypoxic tumourand/or may be a benign tumour or a cancer tumour, for example, a tumourassociated with breast, brain, prostate, colon, esophageal, lung,pancreatic or liver (including hepatocellular carcinoma HCC) cancer. Insome methods of the present invention, the cancer is selected frombreast, brain or prostate cancer. In some methods of the presentinvention, the tumour is a brain tumour.

Metabolic Stability and Relationship with Glucose Metabolism

Without wishing to be bound to any particular theory, the presentinventors believe that the lack of protons alpha to the carbonyl in thelabelled compounds of the invention prevents the compounds from enteringthe citric acid cycle. This is a particular disadvantage associated withthe known PET tracer [¹⁸F]FAC, which undergoes defluorination asdescribed above. Consequently, [¹⁸F]FAC performs inadequately as atracer in the biological imaging of acetate metabolism preclinically in,for example, prostate cancer.

The scheme below shows the first step of the citrate synthase mechanism.The previously published mechanism for entry of acetate into the citricacid cycle¹⁶ has been adapted to show the first step in this cycle for[¹¹C]acetate, [¹⁸F]FAC and ([¹⁸F]3-fluoro-2,2-dimethylpropanoic acid([¹⁸F]FDMP), which is a preferred compound of the present invention. Thepresent inventors believe that the lack of a proton alpha to thecarbonyl prevents Asp375 from attacking compounds of the invention,disabling the compounds from entering the citric acid cycle and losingthe radionuclide label.

In addition to the inability to participate in the citrate acid cycle,the labelled carboxylic acids of the invention may also be less reactivetowards GSH, increasing stability and resistance to metabolism and,where present and applicable, loss of the radionuclide label.

Compounds of the invention have the potential to be converted into anacetyl-CoA derivative through reaction at the carboxylic acid moiety,resulting in the capacity to map the initial step of fatty acidmetabolism. This is thought to be analogous to the use of[¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) to map glucose metabolic flux bytumour cells. However, [¹⁸F]FDG is known to discriminate poorly betweenhealthy and diseased tissue where high glucose uptake/metabolism is afeature of the normal tissue. Compounds of the invention, by contrast,have been shown to demonstrate superior results in these tissues types(vide infra). Accordingly, in some methods of the present invention, thecondition is a tumour located in a tissue-type having high levelsglucose uptake/metabolism, for example a benign or cancerous braintumour.

Steric bulk and inductive effects caused by the necessary alphasubstituents may decrease susceptibility to reaction with GSH. Inpreferred compounds of the invention in which a radionuclide is locatedat a position beta or gamma, preferably beta, to the carbonyl of thecarboxylic acid, the present inventors believe that these compounds maybe less reactive towards GSH and de-labelling due to the increaseddistance of the activating carboxylic acid/carboxylate group. Inespecially preferred compounds of the invention, for example,3-radionuclide-2,2-dimethyl propanoic acids, steric bulk, inductiveeffects and increased distance between the radionuclide and thecarboxylic acid moiety in combination result in compounds particularlyresistant to this reaction pathway.

[¹⁸F]FDMP

[¹⁸F]FDMP has been extensively investigated by the present inventors asa potential PET tracer for cancer imaging and inflammation imaging.Imaging in an inflammation model may be of use in the detection andimaging of non-malignant inflammation related pathologies such asAlzheimer's disease and multiple sclerosis.

[¹⁸F]FDMP Uptake and Trapping

In EMT6 breast cancer cells [¹⁸F]FDMP was converted to an unknownmetabolite; the increased uptake of [¹⁸F]FDMP following incubation withL-carnitine implicates this metabolite as a putative [¹⁸F]FDMP CoA orcarnitine-ester. Extensive work to synthesize the [¹⁹F]FDMP CoA orcarnitine ester reference material was unsuccessful. Using massspectrometry, intracellular levels of [¹⁸F]FDMP were confirmed but againthe inventors were unable to rule out the existence of metabolites bycomparison to [¹⁹F]FAC. Supporting these data, carnitine-esters havepreviously been shown as the major metabolic products of both[¹³C]acetate and [¹³C]propionate catabolism in the heart, measured overa far shorter experimental window of 70 s by hyperpolarized ¹³C MRS—withCoA-esters a minor metabolic product.¹⁵ Based on these data, but withoutwishing to be bound by any particular theory, the present inventorsbelieve that the mechanism of [¹⁸F]FDMA trapping is likely to occur viaCoA and carnitine esters.

Time course biodistribution studies revealed organ-specific variationsin [¹⁸F]FDMP retention and pharmacokinetics, characterized by initialuptake in the liver and clearance through the urinary tract (FIG. 2). Inagreement with previously published data on carnitine-acylcarnitinedistribution,¹⁷ liver-associated [¹⁸F]FDMP rapidly equilibrated with theplasma compartment. The relatively high plasma half-life of [¹⁸F]FDMP,observed by the present inventors, is almost certainly accounted for bythe high reabsorption rate of small chain fatty acids by the proximaltubules,¹⁸ with FDMP and its metabolites excreted into the urine (FIG.5). The distribution profile of [¹⁸F]FDMP in other organs is also inkeeping with known tissue pharmacokinetics of pivalic acid in rodents.¹⁹

The present inventors have further showed that [¹⁸F]FDMP preferentiallyaccumulates in tumours of the breast, brain and prostate. It is thoughtthat upregulation of enzymatic activity (e.g. acetyl CoA synthetase,CRAT), decreased OCTN2 expression or elevated pools of metabolicintermediates may account for increased tumour-to-normal tissueretention. High serum carnitine (˜60 μM in healthy adult males²⁰) mayfurther enhance [¹⁸F]FDMP tumour retention; analogous to the increaseduptake measured in cell experiments following addition of exogenouscarnitine. The present inventors compared tumoural [¹⁸F]FDMP uptake to[¹⁸F]FDG uptake and retention. Although it was impossible todifferentiate between the two radiotracers by normalized uptake valuesin breast adenocarcinoma xenografts (FIG. 7), [¹⁸F]FDMP radiotracerretention was 54% higher in prostate tumours than with [¹⁸F]FDG,indicating the potential utility for [¹⁸F]FDMP for prostate cancerdetection (FIG. 12).

The present inventors further observed that [¹⁸F]FDMP uptake wassignificantly lower than [¹⁸F]FDG in the normal brain. The improvedhuman glioma tumour:brain ratio of 2.5 for [¹⁸F]FDMP, versus 1.3 for[¹⁸F]FDG could be advantageous for [¹⁸F]FDMP visualization of braintumours when compared with [¹⁸F]FDG (FIG. 8). Comparison of [¹⁸F]FDMPwith radiolabeled amino acids [¹¹C]MET, [¹⁸F]FET, and [¹⁸F]DOPA in anorthotopic setting will provide further insight into the effectivenessof [¹⁸F]FDMP for imaging tumours of the brain.

The present inventors have noted that [¹⁸F]FDMP does not appear toprovide an advantage over [¹⁸F]FDG for the differential diagnosis ofcancer versus inflammation (FIG. 13)²¹. [¹⁸F]FDMP and related compoundsmay therefore have utility as radiotracers in non-malignant inflammationrelated pathologies including Alzheimer's disease and multiplesclerosis.

Synthesis

As radionuclides have limited half-life, convenient, reliable and rapidsyntheses for the provision of compositions according to the presentinvention are highly desirable. Accordingly, in one aspect the presentinvention provides methods for the synthesis of radionuclide-labelledcarboxylic acids of the invention and precursors thereof.

Suitable syntheses for some compounds of the present invention may beginwith the corresponding hydroxyl-substituted ester (the hydroxylsubstituent being at the position at which the radionuclide is to beintroduced). The ester may be an optionally substituted alkyl ester oran optionally substituted phenyl or benzyl ester. The radionuclide maybe introduced into this precursor through nucleophilic substitution,preferably through an S_(N)2-type mechanism. Consequently, the hydroxylsubstituent to be displaced is converted to a suitable leaving group,for example, by mesylation or tosylation using methods known in the art.

For example, tosylation may be used to convert a beta-hydroxylsubstituent into a suitable leaving group as detailed below,

wherein R¹, R² and R³ are independently C₁₋₄alkyl, preferably methyl.This leaving group may then be displaced by a suitable radionuclidenucleophile to generate an ester of a radiolabelled nuclide of theinvention. The following scheme shows the reaction for [¹⁸F]fluorine.This example is provided by way of illustration and not by way oflimitation, and any suitable Finkelstein-type reaction may be used tointroduce a appropriate radionuclide label comprising, for example[⁷⁶Br]bromine [¹²³I]iodine, [¹²⁴I]iodine, or [¹²⁵I]iodine.

Suitable [¹⁸F]fluoride nucleophiles include, but are not limited to,kryptand complexes of [¹⁸F]F⁻ formed in the presence of a base. Apreferred kryptand is Kryptofix® K₂₂₂. Suitable bases include potassiumcarbonate and potassium hydrogen carbonate. In some embodiments,potassium hydrogen carbonate may be preferable as a less basicenvironment may disfavour hydrolysis of some ester groups. A furthersuitable [¹⁸F]fluoride nucleophile is [¹⁸F]TBAF, which may be obtainedby combining the generated [¹⁸F]fluoride with a suitabletetrabutylammonium salt, preferably tetrabutyl-ammonium hydrogencarbonate. Analogous to the details provided above, the use oftetrabutylammonium hydrogen carbonate rather than othertetrabutylammonium salts may be preferably in certain embodiments owingto a reduced tendency to undesired ester hydrolysis attributed to thebasicity of the solution. In some methods according to the presentinvention, the step of nucleophilic displacement with a [¹⁸F]fluoridenucleophile is performed at a temperature between 90° C. and 120° C.,preferably about 105° C. and/or lasts for between 5 and 15 minutes,preferably about 10 minutes. The resultant product may be purified usingreverse-phase preparative HPLC (30% ethanol/water) and/or using solidphase extraction using, for example, a C18 cartridge.

Suitable reagents for the inclusion of other radionuclide labels, forexample, [¹¹C]carbon, [¹³N]nitrogen, [¹⁵O]oxygen, or other isotopes suchas [¹³C]carbon may include appropriately labelled organocuprateGrignard-type reagents, amines, and alcohols. For example, to introducea [C^(L)]carbon label a substitution reaction using a suitabledialkylcuprate of general formula R*₂CuLi, wherein R* is[C^(L)]carbon-labelled alkyl group, for example, [¹¹C] or [¹³C]methyl,may be used. [C^(L)]carbon may refer to the naturally most abundantcarbon isotope, [¹²C]carbon or to either a [¹¹C] or [¹³C] carbon asappropriate. As detailed above, the resultant product may be purifiedusing preparative HPLC and/or using solid phase extraction using, forexample, a C18 cartridge, as appropriate.

An alternative approach to the introduction of a radionuclide carbon orcarbon isotope suitable for imaging using MRS or MRI followinghyperpolarisation by DNP into some compounds of the invention may beginwith a suitable acrylate. Use of a dialkylcuprate typically favours thedesired 1,4-addition in preference to 1,2-addition.

To obtain the corresponding labelled carboxylic acids of the invention,the ester moiety is hydrolysed. Hydrolysis may be performed under basicconditions, for example using an aqueous solution of a suitable alkalior alkali earth hydroxide such as sodium hydroxide. In some methods ofthe present invention, the hydrolysis step is performed at a temperaturebetween 50° C. and 70° C., preferably at 60° C. and/or lasts for lessthan 10 minutes, preferably 5 mins.

To prepare suitable compositions, methods of the invention may furthercomprise the step of neutralising the resultant labelled carboxylic acidwith an acid, for example, hydrochloric acid, and a suitable buffer, forexample, a phosphate buffered saline solution, to achieve a compositionof pH suitable for administration. In some methods of the presentinvention, the pH is adjusted to between pH 7 and pH 8, preferablybetween pH 7.2 and pH 7.6, more preferably to about pH 7.4. Somecompositions obtained by methods of the invention have 10% EtOH/PBS orless. Alternatively, in some methods according to the present invention,ethanol may be removed after neutralisation at 40-50° C. under vacuum togive a composition substantially free of ethanol.

Preferably the resulting composition is suitable for injection into asubject without the need for further purification, treatment orprocessing. However, should further purification be required ordesirable, the resulting composition may be purified using solid phaseextraction techniques, for example, using an ion exchange cartridge. Inpreferred embodiments of the present invention in which the label is an[¹⁸F]fluorine and the provision of said composition suitable forinjection into a subject is achieved in less than 120 min from when theaqueous fluoride is delivered and the drying step begins.

As detailed above, it may be desirable to incorporate a carbon isotope,for example, [¹¹C] or [¹³C]carbon, and this may be incorporated at the1-position, that is, at the carboxy carbon. A suitable method for thisincorporation may utilise a Grignard reaction followed by quenching ofthe corresponding anion derived from the bromo-precursor as detailedbelow:

C¹O₂ is [¹¹C]CO₂ or [¹³C]CO₂as appropriate, and R is selected from H, F,C₁₋₄alkyl and OH, wherein the OH may be protected with a suitableprotecting group.

If R is OH or a protected form thereof, the above method may be usefulfor the generation of tosylate precursors suitable for use in thesynthesis detailed above. Alternatively, the above sequence may beperformed when R is fluorine or a suitable radionuclide, for example[¹⁸F]fluorine. Preferred compounds of the invention which may beobtainable using this method include optionally substituted[1-¹¹C]-2,2-C₁₋₄-dialkylpropanoic acids and [1-¹³C]-2,2-C₁₋₄-dialkylpropanoic acids, for example, [1-¹¹C]FDMP, [1-¹³C]FDMP, and[¹⁸F][1-¹³C]FDMP.

The invention will now be further described with reference to thefollowing examples. These are provided as a means of illustration andare not intended to limit the invention.

EXAMPLES

Experimental Procedures

Synthesis of [¹⁸F]FDMP

Methyl 2,2-dimethyl-3-(p-tolylsulfonyloxy)propanoate precursor

Methyl 3-hydroxy-2,2-dimethylpropanoate (193 μL, 1.5 mmol) was dissolvedin dry pyridine (0.5 mL) and DMAP (9.2 mg, 0.075 mmol) in pyridine (0.5mL) was added. Tosyl chloride (347 mg, 1.8 mmol) in pyridine (2 mL) wasthen added and the reaction mixture was stirred at room temperatureunder nitrogen atmosphere for 3h. The reaction was diluted with CH₂Cl₂(30 mL) and water (50 mL). Phases were separated and aqueous layer wasextracted with CH₂Cl₂ (2×30 mL). Combined organic layers were washedwith 1 M HCl (2×50 mL) and brine (50 mL) and dried over Na₂SO₄. The saltwas then filtered off, the reaction mixture concentrated in vacuo andthe residue purified by chromatography on silica gel (15% EtOAc/PE). Thetitle compound was isolated as a white solid (270 mg, 70% yield) ¹H NMR(400 MHz, CDCl₃) δ 7.81 (d, J=8.3 Hz, 2H; Ar), 7.37 (d, J=8.0 Hz, 2H;Ar), 4.03 (s, 2H; 3-H), 3.63 (s, 3H; OMe), 2.48 (s, 3H; Ph-Me), 1.21 (s,6H; CH₃-2); ¹³C NMR (101 MHz, CDCl₃) δ 175.1 (s; CO), 144.9 (s; Ar),132.7 (s; Ar), 129.8 (d, 2C; Ar), 128.0 (d, 2C; Ar), 75.1 (t; C-3), 52.2(q;OCH₃), 42.8 (s; CMe₂), 22.0 (q, 2C; CMe₂), 21.7 (q; Ar-Me); MS[ESI,(%)]: 207 (8, [MH⁺]), 209 (20, [MNa⁺]).

[¹⁸F]-3-fluoro-2,2-dimethyl-propanoic Acid

-   -   Reagent and conditions: a) K₂CO₃, K₂₂₂, [¹⁸F]F⁻ or KHCO₃, K₂₂₂,        [¹⁸F]F⁻ or [¹⁸F]TBAF, 105° C. or 120° C., 10 min.    -   b) NaOH (1M), 60° C., 5 min then HCl (1M) and PBS, or NaOH (1M),        60° C., 5 min then HCl (1M) and 45° C. in vacuo and PBS/a.

a. The methyl 2,2-dimethyl-3-(p-tolylsulfonyloxy)propanoate precursorwas then taken forward for radiochemistry. Initial experiments toincorporate fluorine-18 were carried out using K₂CO₃ and kryptofix toform the K¹⁸F/kryptand complex, followed by addition of the precursor inacetonitrile at 80° C. The reaction was monitored by analytical HPLC.These conditions gave a mixture of radiolabelled products after 5minutes and only 6% product; further heating led to decomposition of theprecursor and only 12% product. The reaction was then undertaken usingDMF as solvent at 105° C., and this showed a robust reaction, giving theproduct (up to 75% of radiolabel incorporation) and [¹⁸F]toluenesulfonylfluoride (25% yield) as a by-product after 15 minutes. At thistemperature the precursor was degraded to give methyl3-hydroxy-2,2-dimethylpropionate. The intermediate was then isolatedusing reverse phase preparative HPLC and 30% ethanol/water as theeluent. The yield of the isolated compound was lower than expected fromthe ratio of conversion. It was suspected that on dilution of thereaction mixture with water, hydrolysis of the methyl ester had occurredgiving the final desired product [¹⁸F]FDMP. The product could not beisolated as it co-eluted at the solvent front with the DMF and anyunreacted fluoride-18. Due to hydrolysis, the isolated yield of themethyl ester intermediate was lower than anticipated (13.9±9.1% decaycorrected, n=7). As a less basic environment should disfavour thehydrolysis of the methyl ester, KHCO₃ was employed instead of K₂CO₃ toform the kryptand complex. The optimal reaction conditions were found tobe heating at 110° C. for 10 min. No other radiochemical peaks apartfrom the desired product was observed. This gave up to 80% ofincorporation of the fluoride as shown by analytical HPLC (40%MeOH/water) and improved the yield of the isolated product to 29.6±19.6%decay corrected (n=6).

Good results were also obtained when [¹⁸F]TBAF was used as source offluoride. The fluoride was dried in the presence of tetrabutylammoniumhydrogen carbonate (TBAHCO₃) to give [¹⁸F]TBAF. The use of the lessbasic TBAHCO₃ led to reduced hydrolysis before preparative HPLC.Analysis of the reaction mixture showed complete incorporation offluoride to give the desired product. After addition of water to thereaction mixture, the preparative HPLC showed 44% intermediate productand 52% of the hydrolysed final product, together with unreactedfluorine-18. The methyl ester protected intermediate was isolated in 37%decay corrected yield.

b. Once isolated from the HPLC eluent, the intermediate product washydrolysed using NaOH (1M) in 5 min at 60° C. which was then neutralisedwith HCl and phosphate buffered saline added to achieve a pH of 7.4 and10% EtOH/PBS or less.

Alternatively, and preferably, after neutralization with HCl, EtOH wasremoved under vacuum at 45° C. and the final solution was buffered withPBS.

In total, the entire synthesis and formulation of [¹⁸F]FDMP from themethyl 2,2-dimethyl-3-(p-tolylsulfonyloxy)propanoate precursor takes 1.5h and delivers [¹⁸F]FDMP ready for injection in EOS of 11.3±4.1% (n=4).

One of the following methods may be used to prepare a dose of acomposition according to the present invention that is suitable foradministration:

Method A. Aqueous [¹⁸F]fluoride was trapped into a QMA cartridge andeluted into a 2 mL Wheaton vial with K₂CO₃ (200 μL of a 12 mg/mL stocksolution) and K222 (800 μL of a 18 mg/mL stock solution). The fluoridewas dried at 120° C. and an azeotrope of MeCN (1 mL) used to aid drying.Methyl 3-tolylsufonyloxy-2,2-dimethyl-propanoate (8 mg) in DMF (300 μL)was added and the reaction mixture was heated at 105° C. for 10 min andthen cooled down to 30° C. using compressed air. The reaction mixturewas quenched with water (700 μL) as labelled intermediate methyl[¹⁸F]-3-fluoro-2,2-dimethyl-propanonate isolated by semipreparative HPLC[Gemini C18 (100×10 mm) column, isocratic 30% EtOH/water method, rt=9min]. NaOH (1M, 200 μL) was added and the mixture heated at 60° C. for 5min, cooled down to room temperature and neutralized with HCl (1M) andPBS to reach neutral pH. Ethanol was removed under vacuum at 45° C., thesolution buffered with PBS and injected without further treatment.

Method B. KHCO₃ (200 μL of a 12 mg/mL stock solution) was used insteadof K₂CO₃ and the labelling carried out as previously described.

Method C. Aqueous [¹⁸F]fluoride was dried in the presence of TBAHCO₃(1.5 M, 22 μL) and the labelling carried out as previously described.

Method D. The radiosynthesis of [¹⁸F]FDMP was automatically performed ona Siemens Explora RN+LC platform. Aqueous [¹⁸F]-fluoride was trappedinto a QMA cartridge preconditioned with water (1 mL) and eluted into a5 mL Wheaton vial with KHCO₃ (100 μL of a 12 mg/mL stock solution inwater) and K222 (400 μL of a 18 mg/mL stock solution in water). Thefluoride was dried at 105° C. and an azeotrope of MeCN (0.5 mL×2) usedto aid drying. Precursor 1 (8 mg) in DMF (450 μL) was added and thereaction mixture was heated at 120° C. for 10 min and then cooled downto 30° C. The reaction mixture was quenched with water (4 mL) andlabeled intermediate [¹⁸F]2 isolated by semipreparative column,isocratic 20% EtOH/water method, flow rate 5 mL/min, retention time(rt)=10 min]. NaOH (1 M, 200 μL) was added and the mixture heated at 60°C. for 5 min then cooled down to 45° C. Ethanol was removed at 45° C.under vacuum for 30 min and the mixture neutralized with HCl (1 M, ˜200μL).

As explained elsewhere, the terms FPIA and FDMP are alternate names forthe same compound.

[¹⁸F]FDMP In Vivo Testing in Mice by PET Imaging

[¹⁸F]FDMP was used in initial experiments by the inventors in healthymice and showed good distribution within the animals and nodefluorination of the tracer, otherwise leading to non-specificretention in bones (PET/CT, FIG. 1). Substantial uptake of [¹⁸F]FDMP wasobserved in the cortex of the kidney, with clearance primarily viaurinary excretion. Tracer localisation was also observed in the heart,liver and intestines.

FIG. 2 shows the biodistribution of [¹⁸F]FDMP in EMT6 murine breastadenocarcinoma xenografts 2, 15, 30 and 60 minutes after administrationas a function of % ID/g (percentage injected dose per gram tissue). Ofparticular note is the low bone uptake which is indicative of minimaldefluorination. FIG. 3 shows the tumour (EMT6) to tissue ratio of[¹⁸F]FDMP 2, 15, 30 and 60 minutes after administration. The data showgood target:background ratio.

Another advantage of [¹⁸F]FDMP is its desirable uptake time (FIG. 4) andstability, in particular, its inability to be a substrate for cellularmetabolism. HPLC chromatrograms of analytes extracted from tissues 30min post [¹⁸F]FDMP injection showed no degradation of FDMP within thebody, besides in urine (FIG. 5). [¹⁸F]FDMP is able to cross theblood-brain barrier (FIG. 6) and, owing to its low background levels,may have utility as an imagining agent for brain tumour detection.[¹⁸F]FDMP compares favourably to [¹⁸F]FDG in vivo. FIG. 7 showscomparable tumour uptake and retention of [¹⁸F]FDMP and [¹⁸F]FDG,confirmed by semi-quantitative parameters derived from the TAC:normalized uptake values at 60 min and values for the tumour area underthe time versus radioactivity curve, while FIG. 8 shows that [¹⁸F]FDMPprovides a superior target:background ratio compared to [¹⁸F]FDG in U87glioma human xenografts.

Cell Culture

EMT6 murine breast cancer cells (LGC Standards) were grown in Waymouth'smedium (Life Technologies), with U87 human glioma cells (LGC Standards)grown in DMEM medium (Life Technologies). DU145 (kind donation from Dr.Almut Schulze, CRUK London Research Institute) and BT474 (LGC Standards)were grown in RPMI (Life Technologies). All media were supplemented with2.5 mL penicillin/streptomycin (10,000 IU·mL⁻¹/10,000 mg·mL⁻¹) and 2 mML-glutamine (Life Technologies). Waymouth's medium contained 15% fetalcalf serum (FCS), with 10% FCS added to DMEM and RPMI. All cells werepropagated at 37° C. in a humidified atmosphere containing 5% CO₂.

Materials

[¹⁸F]FDMP was obtained in end of synthesis yield (EOS) of 7.68±4.99(n=9) in approximately 90 min from aqueous fluoride to formulation.[¹⁸F]FDG was purchased from PETNET solutions (Siemens).

[¹⁸F]FDMP Uptake in Cells

EMT6 cells (2×10⁵) were plated into 6-well plates overnight prior toanalysis. On the day of the experiment fresh growth medium containing0.74 MBq [¹⁸F]FDMP were added to individual wells (1 mL/well). Celluptake was measured over 60 min post radiotracer addition. Plates wereplaced on ice, washed 3 times with ice-cold PBS and lysed in RIPA buffer(Thermo Fisher Scientific Inc.; 1 mL, 10 min). Cell lysates weretransferred to counting tubes and decay-corrected radioactivity wasdetermined on a gamma counter (Cobra II Auto-Gamma counter, PackardBiosciences Co.). Aliquots were snap-frozen and used for proteindetermination following radioactive decay using a BCA 96-well plateassay (Thermo Fisher Scientific Inc.). In addition, 10 μL standards froma 0.74 MBq/mL stock solution were counted to quantitate % radiotraceruptake. For carnitine treatment, cells were incubated with 10 μML-carnitine for the duration of the uptake time course.

Metabolism Experiments in Cells

BT474 cells were seeded in 12-well plates at 2×10⁵ cells per well. Formeasurement of FDMP uptake, the media was replaced with mediasupplemented with 500 μM FDMP and incubated for the indicated times. Allmedia samples were harvested at the same time and immediately dilutedinto ice-cold extraction solution of methanol, acetonitrile, and water(5:3:2) (All chemicals were purchased from Fisher Scientific and wereLC-MS grade). Cell numbers were assumed to be identical. The amount ofextraction solution added to each well was determined by cell countsobtained from a counting plate run in parallel (1 mL/2×10⁶ cells).Extracts were vortexed for 10 min at 4° C. and then centrifuged at16,500 g at 4° C. for 15 min. Supernatants were removed and analyzed byLC-MS (ZIC-pHelic HPLC columns and Exactive Plus Orbitrap MS(ThermoScientific). Data was analyzed using Xcalibur and LCQuan software(ThermoScientific). FDMP and FAC standards were run in isolation forquantification.

In Vivo Tumour Models

All animal experiments were performed by licensed investigators inaccordance with the United Kingdom Home Office Guidance on the Operationof the Animal (Scientific Procedures) Act 1986 and within the publishedguidelines for the welfare and use of animals in cancer research.²² EMT6tumour cells (2×10⁶; 100 μL PBS) were injected subcutaneously on theback of female BALB/c mice (aged 6-8 weeks; Charles River) and grown to˜150 mm³. U87 tumours were grown following subcutaneous injection of5×10⁶ cells (100 μL PBS) on the back of female BALB/c nude mice (aged6-8 weeks; Charles River), with BT474 tumours induced followinginjection of 5×10⁶ cells in matrigel (BD Biosciences; 1:1 ratioPBS-to-matrigel; 100 μL total). Tumour dimensions were measuredperiodically using a caliper and tumour volumes were calculated by theequation: volume=(π/6)×a×b×c, where a, b, and c represent threeorthogonal axes of the tumour. Inflammation was performed in an asepticinflammation model as previously described.²³

In Vivo Radiotracer Stability and Metabolism

Radiolabeled metabolites from plasma and tissues were quantified usingan adapted method.²⁴ BALB/c non-tumour-bearing mice under generalanesthesia (2.5% isofluorane; non-recovery anesthesia) were administereda bolus i.v. injection of [¹⁸F]FDMP (˜7.4 MBq), and sacrificed byexsanguination via cardiac puncture 30 min post radiotracer injection.Heart, urine and liver samples were immediately snap-frozen in liquidnitrogen. Aliquots of heparinized blood were rapidly centrifuged (14000g, 5 min, 4° C.) to obtain plasma. Plasma samples were subsequentlysnap-frozen in liquid nitrogen and kept on dry ice prior to analysis.For analysis, samples were thawed and kept at 4° C. immediately beforeuse. To ice cold plasma and urine (200 μL) was added ice-cold methanol(600 μL) and the resulting suspension centrifuged (14000 g; 4° C.; 3min). 300 μL of the resulting supernatant was added to 1 mL ice coldmobile phase. Samples were filtered through a hydrophilic syringe filter(0.2 μm filter; Millex PTFE filter, Millipore, Mass., USA) and thesample (˜1 mL) then injected via a 1 mL sample loop onto the HPLC foranalysis. Tissues were homogenized in ice-cold methanol (1.5 mL) usingan Ultra-Turrax T-25 homogenizer (IKA Werke GmbH and Co. KG, Staufen,Germany) and subsequently treated as per plasma and urine samples.Samples were analyzed on an Agilent 1100 series HPLC system (AgilentTechnologies, Santa Clara, Calif., USA), using an isocratic method of10% MeOH in an ion pair buffer prepared as follows and a μBondapak C18semi preparative HPLC column (Waters, Milford, Mass., USA; 7.8×3000 mm).Ion pair buffer: 1 mM tetrabutylammonium hydroxide (0.799 g/L) adjustedto pH 7.5 with potassium hydrogen phthalate (0.2 g/L) and sonicated for30 min. Mobile phase was delivered at a flow rate of 3 mL/min.

Biodistribution Studies

[¹⁸F]FDMP (˜3.7 MBq) was injected via the tail vein of anaesthetizedEMT6 tumour-bearing BALB/c mice. The mice were maintained underanesthesia and sacrificed by exsanguination via cardiac puncture at 2,15, 30, 60, 120 or 180 min post radiotracer injection and tissuesharvested. Biodistribution studies with BT474 and U87 tumour-bearingnude mice were performed 60 min post [¹⁸F]FDMP injection. Tissueradioactivity was determined on a gamma counter (Cobra II Auto-Gammacounter, Packard Biosciences Co.) and decay corrected. 10 μL standardsfrom a 1:100 dilution of the stock activity were also counted for datanormalization. Plasma was obtained from terminal blood samples followingcentrifugation (14000 g, 5 min). Data were expressed as percent injecteddose per gram of tissue (% ID/g).

PET Imaging Studies

[¹⁸F]FDMP imaging scans were carried out on a GENISYS⁴ small animal PETscanner (Sofie Biosciences), following a bolus i.v. injection of ˜1.85MBq of the radiotracer into tumour-bearing mice. Static scans wereacquired for 10 min and images were reconstructed usingmaximum-likelihood expectation maximization. In a different cohort ofmice, dynamic PET imaging was performed on a Siemens Inveon PET module,(Siemens Medical Solutions USA) as described in supplementary materials.Dynamic PET imaging was performed for the [¹⁸F]FDMP and [¹⁸F]FDGcomparison study in EMT6 tumour-bearing mice. Static scans on theGENISYS⁴ were used for all other experiments. Siemens Inveon ResearchWorkplace software was used for visualization of radiotracer uptake inthe tumour and to define the three-dimensional volumes of interest(VOI). Tumour radioactivity were normalized to that of the whole body toobtain the normalized uptake value (NUV) to permit comparison of dataobtained using the two scanners.

Dynamic [¹⁸F]FDMP imaging scans were carried outa dedicated small animalPET scanner (Siemens Inveon PET module, Siemens Medical Solutions USA,Inc., Malvern, Pa., USA) following a bolus i.v. injection of ˜3.7 MBq ofthe radiotracer into tumour-bearing mice. Dynamic scans were acquired inlist mode format over 60 min. The acquired data were then sorted into0.5 mm sinogram bins and 19 time frames for image reconstruction (4×15s, 4×60 s, and 11×300 s), which was done by iterative reconstruction(2D-OSEM). The Siemens Inveon Research Workplace software was used forvisualization of radiotracer uptake in the tumour; 30 to 60 mincumulative images of the dynamic data were employed to define3-dimensional (3D) volumes of interest (VOIs). The count densities wereaveraged for all VOIs at each time point to obtain a time versusradioactivity curve (TAC). Tumour TACs were normalized to injected dose,measured by a VDC-304 dose calibrator (Veenstra Instruments, Joure, TheNetherlands), and expressed as percentage injected dose per mL tissue.The area under the TAC, calculated as the integral of % ID/mL from 0-60min, and the normalized uptake of radiotracer at 60 min (% ID/mL60) werealso used for comparisons.

Tumour Cell Uptake and Metabolism

Uptake of [¹⁸F]FDMP into EMT6 tumour cells was linear over the initial30 min of incubation, reaching 1.40±0.10% radioactivity/mg protein. By60 min post radiotracer addition, cell uptake had plateaued at1.47±0.04% radioactivity/mg protein (FIG. 4). [¹⁸F]FDMP uptake increasedby 44% compared to control cells following incubation with 10 μML-carnitine (FIG. 9A) suggesting utilization of carnitine foresterification of [¹⁸F]FDMP. Unlabeled [¹⁹F]FDMP was also rapidly takenup by BT474 cells, detected by mass spectrometry (FIG. 9B). At thehigher probe concentrations used (500 μM in medium), we confirmed thepresence of intracellular [¹⁹F]FDMP; however, no other metabolites wereobserved. The presence of a coenzyme A (CoA) derivative of [¹⁹F]FDMPcould not be ruled out, since in control experiments with[¹⁹F]fluroacetate ([¹⁹F]FAC), only the parent compound and[¹⁹F]fluorocitrate were observed (FIG. 10), which implies conversion viafluoroacetyl-CoA. [¹⁹F]FAC caused negative feedback inhibition in cells(significant increase in citrate and cis-aconitate, together withsignificant decrease in alpha-ketoglutarate due to inhibition ofaconitase; FIG. 10).

Measurements In Vivo

We next evaluated [¹⁸F]FDMP for in vivo tumour imaging. Substantial[¹⁸F]FDMP tumour localization was measured by PET in EMT6 murine breastadenocarcinoma xenografts, clearly visible above background normaltissue (FIG. 7). High [¹⁸F]FDMP uptake was also observed in the bladder,kidney and salivary glands (FIG. 1). Similar to in vitro cell uptake,tumour-associated radioactivity linearly increased over the initial 30min post injection to 9.3±1.0% ID/g, followed by stable retention in thetumour up to 120 min post radiotracer injection (9.8±2.0% ID/g). By 180min, tumour-associated [¹⁸F]FDMP reduced 36% to 6.3±0.7% ID/g. [¹⁸F]FDMPpharmacokinetics were characterized by initial liver uptake, followed byrapid clearance, with the urinary tract being the primary route ofexcretion, determined both by biodistribution (FIG. 2) and from tissueTACs derived from dynamic imaging studies (FIG. 6). Furthermore, tumourradioactivity was comparable for both the murine and human breastadenocarcinoma tumour models, reaching 9.1±0.5% ID/g and 7.6±1.2% ID/gat 60 min post injection in EMT6 and BT474 tumours, respectively (FIG.11).

Radiotracer Stability In Vivo

To confirm that tissue-associated radioactivity corresponded with parent[¹⁸F]FDMP, rather than undesired degradation products, [¹⁸F]FDMPstability was tested in tissues. [¹⁸F]FDMP showed good stability inplasma, liver and heart at 30 min post injection, with only peaks of theparent compound detectable by radioHPLC (FIG. 5). In the urine, theparent peak was the dominant fraction at 30 min (61.8±6.6%radioactivity), with an additional unidentified metabolite peak observedat 6.5 min (37.3±6.0% radioactivity).

Comparison of [¹⁸F]FDMP to [¹⁸F]FDG for Tumour Detection

Given the promising [¹⁸F]FDMP uptake in EMT6 tumours, dynamic [¹⁸F]FDMPPET imaging was performed over 60 min and compared to the gold-standardPET tracer for tumour diagnosis, [¹⁸F]FDG; the same scanner was used forboth radiotracers. High tumour uptake was detected by PET for both[¹⁸F]FDMP and [¹⁸F]FDG in EMT6 tumours, illustrated in 50-60 min images(FIG. 7). In contrast, DU145 prostate adenocarcinoma tumours wereclearly discernable by [¹⁸F]FDMP PET, whereas tumour-associatedradioactivity with [¹⁸F]FDG was not visible above background in 50-60min static scans (FIG. 12). For EMT6 tumours, tumour to whole bodynormalized uptake reached 1.02±0.16 NUV at 60 min for [¹⁸F]FDG, comparedto 1.15±0.13 for [¹⁸F]FDMP (FIG. 12B; P=0.08; n=4-6). Semi-quantitativeimaging parameters derived from the tumour TAC also could notdifferentiate between these two tracers (FIG. 7). Although lower thanfor EMT6 tumours, [¹⁸F]FDMP tumour uptake was 54% higher than [¹⁸F]FDGin DU145 prostate tumours, at 0.73±0.07 and 0.47±0.09 NUV, respectively(FIG. 12B; P=0.002; n=4-5).

Given that [¹⁸F]FDMP passes the blood brain barrier, shown by both gammacounting and by dynamic PET (FIG. 2 and FIG. 6, respectively), we nexttested the potential to use [¹⁸F]FDMP to detect tumours of the brain,comparing uptake values to [¹⁸F]FDG using the well-characterized U87glioma model. In these human-derived xenografts, similarly high[¹⁸F]FDMP and [¹⁸F] FDG tumour uptake was measured 60 minpost-radiotracer injection, at 9.35±1.0% ID/g and 11.0±1.1% ID/g,respectively (FIG. 8; P=0.07; n=4). Although tumour uptake was similarlyhigh for the two radiotracers, [¹⁸F]FDMP provided significantly highertumour/brain ratio of 2.5, compared to just 1.3 with [¹⁸F]FDG (P=0.001;n=4; FIG. 8).

[¹⁸F]FDMP and [¹⁸F]FDG uptake in an orthotopic U87 brain tumour modelwas analysed (FIG. 14). Comparison of the PET images, which wereacquired 15-30 min and 45-60 min post radiotracer injection, show that agood staining of the U87 tumour was visible with FDMP tracer. These datademonstrate that [¹⁸F]FDMP performs well as a tracer for brain tumourmodelling in vivo. The background is very low, so good staining isachieved. The tracer showed a better uptake and staining in PET imagesat an earlier timepoint (15-30 min).

Inflammatory cells are known to display both elevated glycolysis andfatty acid oxidation.²⁷ Given this, we explored [¹⁸F]FDMP and knownability of [¹⁸F]FDG to image this condition using an asepticinflammation model. Both [¹⁸F]FDMP and [¹⁸F]FDG accumulated inchemically-induced inflammatory tissues, at 6.53±1.13% ID/g and5.86±0.68% ID/g, respectively (FIG. 13). There was no significantdifference between inflammation-to-muscle ratios for [^(1B)F]FDMP (2.36)and [¹⁸F]FDG (2.66).

Comparison of [¹⁸F]FDMP to [¹⁸F]FPA

[¹⁸F]2-fluoropropionic acid ([¹⁸F]FPA) has described as a potentiallyuseful PET tracer by Pillarsetty et al.²⁸ The data presented herein showclear advantages for [¹⁸F]FDMP when compared to [¹⁸F] FPA. Datapresented by Pillarsetty show an unfavourable tumour to blood ratio for[¹⁸F]FPA. Essentially, the tumour uptake equals the blood distribution.This suggests that the pharmacokinetic data for [¹⁸F] FPA are lessfavourable than for [¹⁸F]FDMP. [¹⁸F]FDMP's tumour to blood ratio is upto 2.32 after 60 minutes (FIG. 3) vs 1.03 for [¹⁸F]FPA (Table 1 inreference 28).

Furthermore, Pillarsetty et al. describe a tumour-to-brain ratio of 1.27at 60 minutes post-injection for [¹⁸F]FPA, this result being comparableto [¹⁸F]FDG (ratio of 1.3 at 60 minutes as described herein). This isconsiderably worse than the ratio of 2.5 at 60 minutes post injectiondemonstrated by [¹⁸F]FDMP (FIG. 8). In addition, [¹⁸F]FPA lacks the2,2-dialkyl substitution, predicting reduced metabolic stability leadingto loss of the [¹⁸F]label, exemplified by the bone uptake described byPillarsetty et al. (FIG. 4 in reference 28).

Advantages of [¹⁸F]FDMP

[¹⁸F]FDMP is a preferred tracer compound of the invention. Althoughthese advantages are not limited to [¹⁸F]FDMP, and may be similarlyexhibited by other compounds of the invention, [¹⁸F]FDMP has been shownto compare favourably to other PET tracers as shown in the examples.Other tracer compounds for targeting tumours with high fatty acidsynthesis include [¹⁸F]choline and [¹⁸F]fluoroacetate.[¹⁸F]fluoroacetate is not as stable as [¹⁸F]FDMP and therefore may beless interesting for applications such as dual-case PET/CT (2 imagingsessions over a course of time). [¹⁸F]Choline is not recommended forimaging e.g. primary prostate tumours, due to its limited specificity.The results presented herein show that [¹⁸F]FDMP, and other compounds ofthe invention, address an unmet need to provide a stable imaging agentwhich does not undergo de-labelling to lose its radionuclide label, andshows desirable specificity.

[¹⁸F]FDMP was designed by the present inventors for the imaging ofaberrant lipid metabolism associated with malignant transformation. Theradiotracer showed high accumulation in breast, prostate and braintumours, comparable or superior to that of [¹⁸F]FDG. The lower normalbrain uptake (hence inferred superior tumour-to-brain contrast) is apromising characteristic for brain tumour imaging.

Of course, it will be appreciated that the favourable pharmacokineticsdemonstrated for [¹⁸F]FDMP will also be displayed by differentlylabelled isotopologue forms of FDMP, for example, [1-¹³C][¹⁸F]FDMP and[1-¹³C]FDMP.

REFERENCES

The following references are expressly incorporated by reference for allpurposes in their entirety.

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1. A composition comprising a tracer, wherein the tracer is a labelled2,2-di-C₁₋₄-alkylpropanoic acid, 2,2-di-C₁₋₄-alkylbutanoic acid,2,2-di-C₁₋₄-alkylpentanoic acid, or the corresponding carboxylate,wherein the tracer is labelled with: a radionuclide and/or a labelsuitable for polarisation using dynamic nuclear polarisation (DNP). 2.The composition of claim 1, wherein the tracer is aradionuclide-labelled carboxylic acid or the corresponding carboxylate,wherein the radionuclide-labelled carboxylic acid is a compound offormula (I):

wherein Q is a suitable radionuclide; C^(L) is selected from[¹¹C]carbon, [¹²C]carbon, or [¹³C]carbon; n is 1, 2, or 3; R¹ and R² areindependently C₁₋₄-alkyl; and R³ and R⁴ are independently H or F.
 3. Thecomposition of claim 2, wherein R³ and R⁴ are both hydrogen.
 4. Thecomposition of claim 2, wherein n is 1 or 2, optionally wherein n is 1.5. The composition of claim 2, wherein R¹ and R² are both methyl.
 6. Thecomposition of claim 2, wherein the radionuclide Q is selected from[¹¹C]carbon, [¹⁸F]fluorine, [¹³N]nitrogen, [¹⁵O]oxygen, [⁷⁶Br]bromine,[¹²³I]iodine, [¹²⁴I]iodine, and [¹²⁵I]iodine.
 7. The composition ofclaim 1, wherein the radionuclide is [¹⁸F]fluorine.
 8. The compositionof claim 2, wherein C^(L) is [¹²C]carbon.
 9. The composition of claim 1,wherein the carboxylic acid is[1-¹²C][¹⁸F]3-fluoro-2,2-dimethylpropanoic acid.
 10. The composition ofclaim 1, wherein the tracer is a labelled carboxylic acid or thecorresponding carboxylate of formula (Ia):

wherein: R¹ and R² are C₁₋₄alkyl; C^(L) is [¹¹C]carbon, [¹²C]carbon, or[¹³C]carbon; W is R⁵ or Q; R⁵ is H, F, Cl, Br, I, or NH₂; and Q is aradionuclide.
 11. The composition of claim 10, wherein the carboxylicacid is [1-¹³C]2,2-dimethylpropanoic acid, wherein the three position issubstituted with F, [¹⁸F]F, Cl, Br, [⁷⁶Br]Br, I, [¹²³I]I, [¹²⁴I]I,[¹²⁵I]I, or NH₂, optionally with F, [¹⁸F]F, Cl, Br, [⁷⁶Br]Br, [¹²³I]I,[¹²⁴I]I, or [¹²⁵I]I.
 12. The composition of claim 1, wherein thecarboxylic acid is [1-¹³C][¹⁸F]3-fluoro-2,2-dimethylpropanoic acid. 13.The composition of claim 1, wherein the carboxylic acid is[1-¹³C]3-fluoro-2,2-dimethylpropanoic acid.
 14. (canceled)
 15. A methodof imaging for diagnosing a condition in a subject, wherein the methodcomprises: (i) administering the composition of claim 2 to the subject;(ii) detecting gamma rays emitted, either directly or indirectly, by thetracer; (iii) acquiring at least one image associated with the gammarays emitted by the tracer; and (iv) diagnosing the condition in thesubject using the image.
 16. A method of imaging for diagnosing acondition in a subject, wherein the subject has been pre-administeredwith a composition according to claim 1, wherein the method comprises:(i) detecting gamma rays emitted, either directly or indirectly, by thetracer; (ii) acquiring at least one image associated with the gamma raysemitted by the tracer; and (iii) diagnosing the condition in the subjectusing the image.
 17. The method of diagnosis of claim 15, wherein themethod of imaging is positron emission tomography (PET).
 18. The methodof diagnosis of claim 15, wherein Q is [¹²³I] or [¹²⁵I] and the methodof imaging is single-photon emission computed tomography (SPECT).
 19. Amethod of imaging for diagnosing a condition in a subject, wherein themethod comprises: (i) hyperpolarising the tracer compound by dynamicnuclear polarisation (DNP); (ii) administering the composition of claim1 to the subject; (iii) collecting magnetic resonance spectroscopy (MRS)or magnetic resonance imagining (MRI) data associated with thehyperpolarized tracer compound; (iv) acquiring at least one image bymagnetic resonance spectroscopy (MRS) or magnetic resonance imagining(MRI); and (v) diagnosing the condition in the subject using the image.20. A method of imaging for diagnosing a condition in a subject, whereinthe subject has been pre-administered with a composition according toclaim 1, the composition having been hyperpolarised by dynamic nuclearpolarisation (DNP) wherein the method comprises: (i) collecting magneticresonance spectroscopy (MRS) or magnetic resonance imagining (MRI) dataassociated with the hyperpolarized tracer compound; (ii) acquiring atleast one image by magnetic resonance spectroscopy (MRS) or magneticresonance imagining (MRI); and (iii) diagnosing the condition in thesubject using the image.
 21. The method of diagnosis of claim 16,wherein a second imaging technique is used.
 22. The use or thecomposition for use in a method of diagnosis of claim 16, wherein thecondition is a lesion or diseased tissue that has an altered lipidturnover compared to levels in healthy tissue of the same organ ororigin.
 23. The use or the composition for use in a method of diagnosisof claim 16, wherein the condition is a tumour.
 24. The method ofdiagnosis of claim 23, wherein the tumour is a cancer tumour.
 25. Themethod of diagnosis of claim 24, wherein the cancer is breast, brain,prostate, colon, esophageal, lung, pancreatic or liver cancer.
 26. Themethod of diagnosis of claim 24, wherein the cancer is breast, brain, orprostate cancer.
 27. The method of diagnosis of claim 16, wherein thetumour is a hypoxic tumour.
 28. The method of diagnosis of claim 16,wherein the tumour has perturbed carnitine metabolism.
 29. The method ofdiagnosis of claim 16, wherein the condition is metastasis.
 30. Themethod of diagnosis of claim 16, wherein the condition is Alzheimer'sdisease.
 31. The method of diagnosis of claim 16, wherein the conditionis multiple sclerosis.
 32. The method of diagnosis of claim 16, whereinthe condition is a heart-related disease or disorder.
 33. The method ofdiagnosis of claim 16, wherein the condition is a genetic/epigeneticdisease of lipid metabolism.
 34. A method for imaging a condition in asubject using a composition according to claim 1, the method comprisingthe steps of: (i) administering a composition comprising a traceraccording to claim 1 to the subject; (ii) detecting gamma rays emittedby the tracer; and (iii) acquiring at least one image associated withthe gamma rays emitted by the tracer.
 35. A method for detecting acondition in a subject the subject having been pre-administered with acomposition according to claim 1, the method comprising the steps of:(i) detecting gamma rays emitted, either directly or indirectly, by thetracer; and (ii) evaluating the condition based on the detection of thegamma rays emitted by the tracer.
 36. The method of claim 34, whereinthe method further comprises the step of diagnosing a condition in thesubject using the image.
 37. The method of claim 34, wherein the methodfurther comprises the step of comparing the image with a previouslyobtained image to monitor progression of the condition and/or responseto therapy.
 38. A method of synthesising a composition comprising acompound of formula (II):

the method comprising the step of treating a compound of formula (III)

with a reagent comprising a radionuclide nucleophile to give a compoundof formula (IV)

wherein Q is a radionuclide or group comprising a radionuclide; C^(L) isselected from [¹²C]carbon or [¹³C]carbon; and R¹, R² and R³ areindependently C₁₋₄alkyl.
 39. The method of claim 38, wherein each of R¹,R² and R³ is methyl.
 40. The method of claim 38, wherein theradionuclide nucleophile is [¹⁸F]fluoride.
 41. The method of claim 40,wherein the reagent comprising an [¹⁸F]fluoride nucleophile is selectedfrom: (i) K₂CO₃, K₂₂₂, [¹⁸F]F ; (ii) KHCO₃, K₂₂₂, [¹⁸F]F⁻; or (iii)[¹⁸F]TBAF.
 42. The method of claim 41, wherein the reagent comprising an[¹⁸F]fluoride nucleophile is [¹⁸F]TBAF.
 43. The method of claim 38,wherein the step of treating a compound of formula (III) with a reagentcomprising an [¹⁸F]fluoride nucleophile is performed at a temperaturebetween 90° C. and 120° C. and/or lasts for between 5 and 15 minutes.44. The method of claim 38, the method further comprising purificationof the compound of formula (IV) using a) preparative HPLC, and/or b)solid phase extraction using a C18 cartridge.
 45. The method of claim38, the method further comprising the step of hydrolysing the compoundof formula (IV) with sodium hydroxide to unmask the carboxylic acidmoiety.
 46. The method of claim 45, wherein the step of hydrolysing thecompound of formula (IV) is performed at a temperature between 50° C.and 70° C. and/or lasts for less than 10 minutes.
 47. The method ofclaim 40, the method further comprising the step of neutralising theresultant [¹⁸F]fluorinated carboxylic acid/carboxylate with hydrochloricacid and a phosphate buffered saline solution to achieve a pH of 7.4 and10% EtOH/PBS or less.
 48. The method of synthesis of claim 38, whereinthe composition comprising a compound of formula (II) is obtained inless than 120 min from the beginning of step of treating a compound offormula (III) with a reagent comprising an [¹⁸F]fluoride nucleophile.49. A kit for the preparation of a radionuclide-labelled imaging agent,the kit comprising a compound of formula (III)

and instructions for a method according to claim
 38. 50. The method ofclaim 35, wherein the method further comprises the step of diagnosing acondition in the subject using the image.
 51. The method of claim 35,wherein the method further comprises the step of comparing the imagewith a previously obtained image to monitor progression of the conditionand/or response to therapy.